Chemiluminescent acridinium esters have emerged to be extremely useful labels for both immunoassays as well as nucleic acid assays. Light emission from acridinium esters is triggered by the reaction with alkaline hydrogen peroxide which causes scission of the phenolic ester bond and the formation of excited state acridone, which is the light emitting species. The formation of a dioxetanone intermediate following the departure of the phenol leaving group has been proposed in the reaction mechanism and is presumed to be the precursor to the excited state acridone. The duration of light emission from acridinium esters which, depends upon the reaction conditions, is normally complete within a few seconds and can be quantitatively measured using a luminometer. The application of the acridinium ester 9-carboxyphenyl-N-methylacridinium bromide in an immunoassay was disclosed by Simpson, J. S. A. et al. (Nature vol. 279, pp. 646-647 (1979). However, this acridinium ester is quite unstable, thereby limiting its commercial utility. This instability arises from hydrolysis of the labile 9-carboxyphenyl ester linkage between the phenol and the acridinium ring.
In an attempt to stabilize the phenolic ester in acridinium esters, Law et al. (Journal of Bioluminescence and Chemiluminescence, vol. 4, pp. 88-89 (1989) introduced two, flanking methyl groups to stabilize this ester bond. The resulting sterically stabilized acridinium ester, abbreviated as DMAE-NHS [2′,6′-dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl 10-methylacridinium-9-carboxylate] was found to have the same light output as an acridinium ester lacking the two methyl groups. On the other hand, the stability of the former compound when conjugated to an immunoglobulin was vastly superior and showed no loss of chemiluminescent activity even after one week at 37° C. at pH 7. In contrast, the unsubstituted acridinium ester only retained 10% of its activity when subjected to the same treatment.
To alleviate the hydrophobic nature of DMAE and its derivatives, U.S. Pat. No. 5,656,426 by Law et al. disclosed a hydrophilic version of DMAE termed NSP-DMAE-NHS ester where the N-methyl group had been replaced with an N-sulfopropyl (NSP) group. Both DMAE and NSP-DMAE are now widely used in the commercial, automated immunoassay analyzers ACS:180™ and Centaur™ of Siemens Healthcare Diagnostics.
To further increase the hydrophilicity of NSP-DMAE, Natrajan et al. in U.S. Pat. No. 6,664,043 B2 disclosed NSP-DMAE derivatives with hydrophilic modifiers such as poly(ethylene)glycol and anionic spermine derivatives attached to the phenol. The structure of one such acridinium ester called NSP-DMAE-HEG-Glutarate-NHS, (abbreviated as NSP-DMAE-HEG) and containing a hexa(ethylene)glycol derivative (HEG) is illustrated in the following drawing. In this compound an α,ω-diamino hexa(ethylene) glycol (diamino-HEG) moiety is attached to the phenol to increase the aqueous solubility of the acridinium ester. A glutarate moiety was appended to the end of diamino-HEG and was converted to the NHS ester to enable labeling of various molecules. In addition to HEG, anionic modifiers derived from spermine with sulfonate and carboxylate groups were also disclosed by Natrajan et al. in U.S. Pat. No. 6,664,043 B2.
Recently, Natrajan et al. in U.S. Pat. No. 7,309,615 B2 described hydrophilic, high quantum yield acridinium compounds containing hydrophilic alkoxy groups (OR*) at C2 and/or C7 of the acridinium ring, wherein R* is a group comprising a sulfopropyl moiety or ethylene glycol moieties or a combination thereof. The enhanced light output from such compounds and their hydrophilic nature made them useful for improving the sensitivity of immunoassays. The structure of once such compound called NSP-2,7-(OMHEG)2-DMAE-AC-NHS (abbreviated as HQYAE) is illustrated below. This compound and other compounds described in U.S. Pat. No. 7,309,615 B2 contained poly(ethylene)glycol or anionic sulfonate groups attached to the acridinium ring instead of the phenol.

The incorporation of poly(ethylene)glycol (PEG) moieties and anions in the structures of acridinium esters as disclosed in the aforementioned patents were meant to increase the hydrophilic nature of these acridinium esters as well as to lower the non-specific binding of conjugates derived from these compounds. Non-specific binding, in assays using solid phases such as particles or microtiter plates, are undesired binding interactions of these acridinium ester conjugates to these solid phases. These undesired binding interactions typically increase the background of the assay leading to a net lowering of the signal to background ratio in the assay and thereby decreasing assay sensitivity.
The use of poly(ethylene)glycol to devise inert surfaces that resist protein adsorption has been described in the prior art. For example, Ostuni et al. in J. Am. Chem. Soc. 2000, 17, 5605-5620, evaluated numerous functional groups attached to self-assembled monolayers for resistance to protein adsorption and observed that poly(ethylene)glycol functional groups conferred the best resistance. More recently, Ladd et al. in Biomacromolecules 2008, 9, 1357-1361; and others have reported that zwitterion-modified hydrophilic surfaces are as inert to protein adsorption as PEG and moreover, it has been postulated that zwitterions would be chemically more stable than PEG owing to the latter's propensity for oxidative cleavage. Structures of some common zwitterions such as sulfobetaines, carboxybetaines, phosphobetaines and amine oxides are illustrated in the following drawing. Like PEG, these zwitterions are normally electrically neutral, because of the balance of positive and negative charges within a given structure (R1-R4 are typically alkyl groups).
Examples of Zwitterions

Sulfobetaines where the nitrogen atom is quarternary maintain their electrical neutrality over a wide pH range. On the other hand sulfobetaines with a trisubstituted nitrogen can acquire a negative charge at higher pH (pH>7) because they can be deprotonated at the nitrogen thereby neutralizing its positive charge. Similarly, carboxybetaines are electrically neutral at higher pH but can be protonated at the carboxylate group and acquire a net positive charge at low pH (pH<5).
Zwitterion-modified fluorescent dyes for applications in proteomics have recently been reported by Dratz and Grieco in US Patent Application No. 0259333 A1 (November 2007). Proteins labeled with zwitterionic dyes were observed to be water soluble and more amenable for electrophoretic analysis and the above authors have claimed methods for protein analysis using zwitterionic dyes.
The incorporation of electrically neutral, zwitterionic functional groups in chemiluminescent acridinium esters to improve their properties such as non-specific binding has not been described the prior art. Such compounds complement the PEG-containing acridinium esters described by Natrajan et al. in U.S. Pat. No. 6,664,043 B2 and U.S. Pat. No. 7,309,615 B2 and, have the potential to improve assay sensitivity.
There is a continuing need in the art for improved chemiluminescent tags for immunoassays and the like. It is therefore an object of the invention to provide chemiluminescent acridinium compounds which exhibit reduced non-specific binding to a solid phase.